Abstract One of the stumbling blocks to development of a live-attenuated RSV vaccine is induction of long-lasting immunity, which does not occur even with wild-type RSV infection. Since induction of robust adaptive immunity responses requires a strong innate immune activation, the short-lived anti-RSV response may be due to viral inhibition of type I interferon (IFN) induction. Antagonism of IFN production is largely due to the RSV NS1 protein, which is not absolutely essential for virus replication in culture although deletion of NS1 results in decreased viral replication even in IFN-deficient cells such as Vero. This proposal will identify domains and/or specific residues of NS1 responsible for IFN antagonism by targeted mutagenesis. Mutants that ablate the IFN antagonist function without affecting protein stability will be inserted into recombinant RSV (rRSV) and tested for their capacity to replicate in Vero cells and induce IFN in A549 cells. NS1 mutant rRSV that induce high levels of IFN expression and replicate well in these cell lines will be tested for IFN antagonism and replication in human airway epithelial (HAE) cultures. Further, the NS1 mutant rRSV will be tested in cotton rats, which are the best small animal model for RSV infection to determine replication in vivo, pathogenicity, immunogenicity, and protection from WT challenge. The best candidate NS1 mutants will be combined with the `repaired' G gene (Project 2) and the best attenuating L methyltransferase (MTase) mutations (Project 3) to produce double and triple mutant viruses that replicate well in Vero cells, induce high levels of IFN, efficiently infect HAE culture, and are attenuated and protective in cotton rats. Although beyond the scope of this P01, these vaccine candidates will be tested in non-human primate trials in the future.